Disulfides in proteins play an important role in the maintenance of biological activity and conformational stability. It is possible to produce peptides and proteins having the correct sequence of amino acid residues, by solution and solid phase synthesis. However, unless the peptides can be formed into the correct spatial configuration, i.e. fold correctly, the product will usually be physiologically deficient. A critical step in the folding of proteins is the formation of disulfide bonds from cysteine residues. This is an oxidative step in which sulfhydryl groups are converted to disulfide bonds, thereby forming bridges which assist in folding the peptide or protein into a desired configuration.
Among the conventional methods for the formation of disulfide bonds, air oxidation in aqueous medium is the most commonly used (1-3). Air oxidation usually requires a long duration at basic or neutral pH for completion and a high dilution of peptide or protein concentration to be effective. Nevertheless, it enjoys an advantage that it produces water as a harmless byproduct of the reaction. A variation of the air oxidation method is the thiol-disulfide interchange reaction using a mixture of reduced and oxidized glutathiones (3). The mixed disulfide interchange method is usually effective in the basic pH range. Because the air oxidation and the mixed disulfide interchange method are slow processes, they allow equilibrations of different conformers to produce thermodynamic controlled products. In contrast, strong oxidizing agents such as I.sub.2 and K.sub.3 Fe(CN).sub.6 that produce kinetic-controlled products are often used for simple peptides containing only a single disulfide bond (4-5). These sulfur-sulfur forming agents are such powerful oxidants that the oxidations are usually performed under carefully controlled conditions to prevent over oxidation. They have the advantage of being applicable in the acidic range, but suffer from the limitation that byproducts generated usually require extensive purification procedures. Several nucleophilic amino acids such as Met, Tyr, Trp, and His are particularly susceptible to these strong oxidants (6). Because of these limitations, strong oxidizing agents are seldom used for the simultaneous refolding and disulfide formation of multi-disulfide bonded peptides or proteins.
Despite these disadvantages, the oxidation methods using air or mixed disulfides are useful for many syntheses, particularly for those involving acidic peptides or proteins (7). However, for basic and hydrophobic peptides that tend to aggregate and precipitate out of solution at or near their basic or neutral isoelectric points during the folding process, the air or mixed disulfide methods for oxidation are not satisfactory. It has been observed that this is the case in the synthesis of several basic and hydrophobic disulfide-rich peptides. In the synthesis of a series of viral growth factors (8-9), the disulfide formation by air oxidation or mixed disulfide method produced precipitation even in the presence of a strong denaturant such as 6M urea. The precipitation resulted in unacceptably low yields of the desired product.
It is, therefore, highly desirable to devise a new method for disulfide formation that is similar in mildness to air oxidation but can be conducted under acidic conditions at an efficient rate with no harmful byproducts.